Signaling by the ERK-MAP kinases and RSK family of protein kinases plays a significant role in the regulation of processes controlling cell growth and proliferation, cell migration and invasion, cell survival, and differentiation. Thus, when improperly regulated, ERK/RSK signaling also contribute to the neoplastic state. Indeed, several oncogenes and their cellular homologs have been found to directly participate in the regulation of ERK/RSK signaling and well over 50% of human cancers have this pathway activated. Therefore, it is critical to thoroughly define ERK/RSK regulatory mechanisms and their downstream effectors. The original goals during the last funding period (10 year MERIT award) were to characterize RSK regulation by ERK and other inputs, identify RSK interacting proteins and identify new RSK targets. The ultimate goal was to define the molecular and biochemical basis of ERK and RSK activation, downstream signaling and how these events regulate various biological processes including transcriptional and translational control, cell migration and cell survival. These goals evolved to include characterization of ERK signaling via distinct interaction motifs and the biological consequences. We have made several significant discoveries that continue to further our understanding of the regulation and functional consequences of ERK/RSK signaling in normal health and when improperly regulated, diseases such as cancer. Our previous work and that of others sets the foundation for the current proposal. From our innovative approaches and proposed research, we will define the ERK/RSK phosphoproteome and proteome using a unique ERK signaling system we have developed and a reductive dimethylation/mass spectrometry approach (aim 1). The initial data have already been obtained after acute EGF stimulation and this is to be expanded so that we will define the changing proteome/phosphoproteome with a time course analysis to inform us as to how cells make specific cell fate decisions. Aim 1 also includes gene expression analysis during this time course to complement the mass spectrometry, and has already revealed genes specific to ERK/RSK-regulated cell proliferation vs. genes specific to ERK-regulated epithelial to mesenchymal transition. Aim 2 will use data from the screen to initiate our efforts to define cross-talk pathways and negative feedback loops regulated by RSK that contribute to signaling and cell fate decisions. Phosphoproteomic data and published results will also be used to characterize signaling linking RSK to cell migration and survival via regulation of the actomyosin cytoskeleton (aim 3). Through these aims, we will identify and characterize new upstream regulators and downstream effectors of ERK/RSK, further explaining how improper regulation of the Ras pathway contributes to a variety of human diseases. Furthermore, our research will result in the identification of new biomarkers and potential therapeutic targets needed for detection and intervention in diseases resulting from improper ERK/RSK signaling.